Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the ß-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.

Exploring the uncharted territory of the potential protein-protein interactions of cytosolic malate dehydrogenase.

In this review, we examine the protein-protein interactions of cytosolic malate dehydrogenase (MDH), an under-studied area in cellular metabolism. We provide a comprehensive overview of MDH involvement in metabolism, especially its interactions with metabolic partners and dynamics of changing metabolism. We present an analysis of the biophysical nature of these interactions and the current methods used to study them. Our review includes an assessment of computational docking studies, which offer initial hypotheses about potential MDH interaction partners. Furthermore, we provide a summary of the sparse yet insightful experimental evidence available, establishing a foundation for future research. By integrating biophysical analysis and methodological advancements, this paper aims to illuminate the intricate network of interactions involving cytosolic MDH and their metabolic implications. This work not only contributes to our understanding of MDH's role in metabolism but also highlights the potential impact of these interactions in metabolic disorders.

20(S)-protopanaxadiol-aglycone downregulation of the full-length and splice variants of androgen receptor.

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

The Evolving Landscape of Therapeutics for Epilepsy in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a rare multisystem genetic disorder characterized by benign tumor growth in multiple organs, including the brain, kidneys, heart, eyes, lungs, and skin. Pathogenesis stems from mutations in either the TSC1 or TSC2 gene, which encode the proteins hamartin and tuberin, respectively. These proteins form a complex that inhibits the mTOR pathway, a critical regulator of cell growth and proliferation. Disruption of the tuberin-hamartin complex leads to overactivation of mTOR signaling and uncontrolled cell growth, resulting in hamartoma formation. Neurological manifestations are common in TSC, with epilepsy developing in up to 90% of patients. Seizures tend to be refractory to medical treatment with anti-seizure medications. Infantile spasms and focal seizures are the predominant seizure types, often arising in early childhood. Drug-resistant epilepsy contributes significantly to morbidity and mortality. This review provides a comprehensive overview of the current state of knowledge regarding the pathogenesis, clinical manifestations, and treatment approaches for epilepsy and other neurological features of TSC. While narrative reviews on TSC exist, this review uniquely synthesizes key advancements across the areas of TSC neuropathology, conventional and emerging pharmacological therapies, and targeted treatments. The review is narrative in nature, without any date restrictions, and summarizes the most relevant literature on the neurological aspects and management of TSC. By consolidating the current understanding of TSC neurobiology and evidence-based treatment strategies, this review provides an invaluable reference that highlights progress made while also emphasizing areas requiring further research to optimize care and outcomes for TSC patients.

Removal of estrogens and estrogenicity through drinking water treatment.

Estrogenic compounds have been shown to be present in surface waters, leading to concerns over their possible presence in finished drinking waters. In this work, two in vitro human cell line bioassays for estrogenicity were used to evaluate the removal of estrogens through conventional drinking water treatment using a natural water. Bench-scale studies utilizing chlorine, alum coagulation, ferric chloride coagulation, and powdered activated carbon (PAC) were conducted using Ohio River water spiked with three estrogens, 17ß-estradiol, 17α-ethynylestradiol, and estriol. Treatment of the estrogens with chlorine, either alone or with coagulant, resulted in approximately 98% reductions in the concentrations of the parent estrogens, accompanied by formation of by-products. The MVLN reporter gene and MCF-7 cell proliferation assays were used to characterize the estrogenic activity of the water before and after treatment. The observed estrogenic activities of the chlorinated samples showed that estrogenicity of the water was reduced commensurate with removal of the parent estrogen. Therefore, the estrogen chlorination by-products did not contribute appreciably to the estrogenic activity of the water. Coagulation alone did not result in significant removals of the estrogens. However, addition of PAC, at a typical drinking water plant dose, resulted in removals ranging from approximately 20 to 80%.

Covalent and Noncovalent Loading of Doxorubicin by Folic Acid-Carbon Dot Nanoparticles for Cancer Theranostics.

With special properties such as excellent fluoresce features, low toxicity, good biocompatibility, permeability, and easy clearance from the body, carbon dot (CD)-based nanoparticles (NPs) have the potential to deliver drugs and use in vivo diagnostics through molecular imaging. In this work, folic acid-CD (FA-CD) NPs were prepared to deliver doxorubicin (Dox) covalently and noncovalently as cancer theranostics. FA was conjugated to the surface of CDs for targeting cancer cells with overexpressing folate receptors. CDs prepared with various amounts of precursors lead to their associated NPs with different photoluminescence properties and drug release profiles. The loading of Dox and its releasing data depends on the linkage of drug Dox to FA-CD and CD composition. All NPs were characterized by UV-vis, Fourier transform infrared spectroscopy, and dynamic light scattering. The noncovalent FA-CD-Dox NPs were preferred with a simple preparation process, excellent photoluminescence, and in vitro drug release properties. The noncovalent FA-CD-Dox showed the best efficacy against MDA-MB-231 compared to the CD-Dox and covalent FA-CD-Dox.

Efficacy and safety of balovaptan for socialisation and communication difficulties in autistic adults in North America and Europe: a phase 3, randomised, placebo-controlled trial.

BACKGROUND: There are no approved pharmacological therapies to support treatment of the core communication and socialisation difficulties associated with autism spectrum disorder in adults. We aimed to assess the efficacy, safety, and pharmacokinetics of balovaptan, a vasopressin 1a receptor antagonist, versus placebo in autistic adults. METHODS: V1aduct was a phase 3, randomised, placebo-controlled, double-blind trial, conducted at 46 sites across six countries (the USA, the UK, France, Italy, Spain, and Canada). Eligible participants were aged 18 years or older with an intelligence quotient (IQ) of 70 or higher, and met the criteria for moderate-to-severe autism spectrum disorder (DSM-5 and Autism Diagnostic Observation Schedule). Participants were randomly allocated (1:1), with an independent interactive voice or web-based response system, to receive balovaptan (10 mg) or placebo daily for 24 weeks. Randomisation was stratified by an individual's baseline Vineland-II two-domain composite (2DC) score (<60 or ≥60), sex, region (North America or rest of world), and age (<25 years or ≥25 years). Participants, study site personnel, and the sponsor were masked to treatment assignment. The primary endpoint was change from baseline in Vineland-II 2DC score (the mean composite score across the Vineland-II socialisation and communication domains) at week 24. The primary analysis was done with ANCOVA in the intention-to-treat population. The V1aduct study was terminated for futility after around 50% of participants completed the week 24 visit. This trial is registered with ClinicalTrials.gov (NCT03504917). FINDINGS: Between Aug 8, 2018, and July 1, 2020, 540 people were screened for eligibility, of whom 322 were allocated to receive balovaptan (164 [51%]) or placebo (158 [49%]). One participant from the balovaptan group was not treated before trial termination and was excluded from the analysis. 60 participants in the balovaptan group and 55 in the placebo group discontinued treatment before week 24. The sample consisted of 64 (20%) women and 257 (80%) men, with 260 (81%) participants from North America and 61 (19%) from Europe. At baseline, mean age was 27·6 years (SD 9·7) and mean IQ score was 104·8 (18·1). Two (1%) participants were American Indian or Alaska Native, eight (2%) were Asian, 15 (5%) were Black or African American, 283 (88%) were White, four (1%) were of multiple races, and nine (3%) were of unknown race. Mean baseline Vineland-II 2DC scores were 67·2 (SD 15·3) in the balovaptan group and 66·2 (17·7) in the placebo group. The interim futility analysis showed no improvement for balovaptan versus placebo in terms of Vineland-II 2DC score at week 24 compared with baseline, with a least-squares mean change of 2·91 (SE 1·52) in the balovaptan group (n=79) and 4·75 (1·60) in the placebo group (n=71; estimated treatment difference -1·84 [95% CI -5·15 to 1·48]). In the final analysis, mean change from baseline in Vineland-II 2DC score at week 24 was 4·56 (SD 10·85) in the balovaptan group (n=111) and 6·83 (12·18) in the placebo group (n=99). Balovaptan was well tolerated, with similar proportions of participants with at least one adverse event in the balovaptan group (98 [60%] of 163) and placebo group (104 [66%] of 158). The most common adverse events were nasopharyngitis (14 [9%] in the balovaptan group and 19 [12%] in the placebo group), diarrhoea (11 [7%] and 14 [9%]), upper respiratory tract infection (ten [6%] and nine [6%]), insomnia (five [3%] and eight [5%]), oropharyngeal pain (five [3%] and eight [5%]), and dizziness (two [1%] and ten [6%]). Serious adverse events were reported for two (1%) participants in the balovaptan group (one each of suicidal ideation and schizoaffective disorder), and five (3%) participants in the placebo group (one each of suicidal ideation, panic disorder, limb abscess, urosepsis, colitis [in the same participant with urosepsis], and death by suicide). No treatment-related deaths occurred. INTERPRETATION: Balovaptan did not improve social communication in autistic adults. This study provides insights into challenges facing autism spectrum disorder trials, including the considerable placebo response and the selection of appropriate outcome measures. FUNDING: F Hoffmann-La Roche.

Building a Diverse Workforce and Thinkforce to Reduce Health Disparities.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

Glyceollin I, a novel antiestrogenic phytoalexin isolated from activated soy.

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II, and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogen-stimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human ERalpha and in an estrogen-responsive element-based luciferase reporter assay. We identified glyceollin I as the active component of the combined glyceollin mixture. Ligand-receptor modeling (docking) of glyceollin I, II, and III within the ERalpha ligand binding cavity demonstrates a unique type II antiestrogenic confirmation adopted by glyceollin I but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17beta-estradiol-stimulated expression of progesterone receptor and stromal derived factor-1alpha. Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormone-dependent tumors.

A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus.

Previous studies have suggested that the lactate dehydrogenase-B gene (Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5'-GATGTG-3' spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner.

Proteomic analysis of tumor necrosis factor-alpha resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype.

INTRODUCTION: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-alpha. Additionally, the relationship between TNF-alpha resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated. METHODS: To compare differences in the proteome of the TNF-alpha resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-alpha resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT. RESULTS: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (delta-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression. CONCLUSIONS: This study demonstrates that MEK5 over-expression promotes a TNF-alpha resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

ZB716, a steroidal selective estrogen receptor degrader (SERD), is orally efficacious in blocking tumor growth in mouse xenograft models.

Advances in oral SERDs development so far have been confined to nonsteroidal molecules such as those containing a cinnamic acid moiety, which are in earlystage clinical evaluation. ZB716 was previously reported as an orally bioavailable SERD structurally analogous to fulvestrant. In this study, we examined the binding details of ZB716 to the estrogen receptor alpha (ERα) by computer modeling to reveal its interactions with the ligand binding domain as a steroidal molecule. We also found that ZB716 modulates ERα-coregulator interactions in nearly identical manner to fulvestrant. The ability of ZB716 to inhibit cell growth and downregulate ER expression in endocrine resistant, ERα mutant breast cancer cells was demonstrated. Moreover, in both the MCF-7 xenograft and a patient derived xenograft model, orally administered ZB716 showed superior efficacy in blocking tumor growth when compared to fulvestrant. Importantly, such enhanced efficacy of ZB716 was shown to be attributable to its markedly higher bioavailability, as evidenced in the final plasma and tumor tissue concentrations of ZB716 in mice where drug concentrations were found significantly higher than in the fulvestrant treatment group.

Rational Design of a Boron-Modified Triphenylethylene (GLL398) as an Oral Selective Estrogen Receptor Downregulator.

Development of orally bioavailable nonsteroidal selective estrogen receptor downregulators (SERDs) provides clinical opportunities for the long-term treatment and adjuvant therapy of breast cancer at all stages. We describe the design, synthesis, and identification of a boron-modified GW7604 derivative (GLL398, 9), a SERD candidate, in which a boronic acid functional group replaces the phenolic hydroxyl group of GW7604. Compound 9 strongly binds to ERα in a fluorescence resonance energy transfer binding assay (IC50 = 1.14 nM) and potently degrades ERα in MCF-7 breast cancer cells (IC50 = 0.21 µM). Most importantly, the introduction of the boronic acid group confers superior oral bioavailability of 9 (AUC = 36.9 µg·h/mL) in rats as compared to GW7604 (AUC = 3.35 µg·h/mL). The strikingly favorable pharmacokinetic property of 9 makes it a promising oral SERD suitable for clinical evaluation.

Aptamer-functionalized hybrid nanoparticle for the treatment of breast cancer.

PURPOSE: Resistance to chemotherapeutic agents such as doxorubicin is a major reason for cancer treatment failure. At present the treatment option for metastatic breast cancer is very poor. Therefore, development of an effective therapeutic strategy to circumvent MDR of metastatic breast cancer is highly anticipated. The MDR of metastatic breast cancer cells was accompanied with the overexpression of P-gp transporter. Even though the overexpression of P-gp could be minimized by silencing with siRNA, the question is how they can be selectively targeted to the cancer cells. We propose that aptamer surface labeling of the nanoparticles could enhance the selectively delivery of p-gp siRNA into the metastatic breast cancer cells. Our hypothesis is that conjugating nanoparticles with a cancer cell specific aptamer should allow selective delivery of therapeutic drugs to tumor cells leading to enhanced cellular toxicity and antitumor effect as compared to unconjugated nanoparticles. The primary objective of this study is to develop a targeted nanocarrier delivery system for siRNA into breast cancer cells. DESIGN METHODS: For targeted delivery, Aptamer A6 has been used which can bind to Her-2 receptors on breast cancer cells. For aptamer binding to particle surface, maleimide-terminated PEG-DSPE (Mal-PEG) was incorporated into the nanoparticles. Initially, three blank hybrid nanoparticles (i.e. F21, F31, and F40) out of nine different formulations prepared by high pressure homogenization (HPH) using different amount of DOTAP, cholesterol, PLGA or PLGA-PEG and Mal-PEG were chosen. Then protamine sulfate-condensed GAPDH siRNA (TRITC conjugated; red) or P-gp siRNA was encapsulated into those nanoparticles. Finally, the particles were incubated with aptamer A6 (FITC conjugated; green) for surface labeling. RESULTS: Aptamer labeled-nanoparticles having PLGA are smaller in size than those having PLGA-PEG. Surface charge was reduced when the particles were labeled with aptamer. Cell transfection was increased significantly in Her-2 (+) SKBR-3 and 4T1-R cells but not in Her-2 poorly expressed MDA MB-231 and MCF-7 cells. The knockdown of P-gp was increased significantly when the particles were labeled with aptamer. No significant cellular toxicity was observed for any of these formulations. CONCLUSION: This preliminary study concludes that aptamer-functionalized hybrid nanoparticles could be used to deliver P-gp targeted siRNA into the breast cancer cells to overcome chemoresistance.

Resibufogenin corrects hypertension in a rat model of human preeclampsia.

The study of the pathogenesis of preeclampsia has been hampered by a relative dearth of animal models. We developed a rat model of preeclampsia in which the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), is elevated. These animals develop hypertension, proteinuria, and intrauterine growth restriction. The administration of a congener of MBG, resibufogenin (RBG), reduces blood pressure to normal in these animals, as is the case when given to pregnant animals rendered hypertensive by the administration of MBG. Studies of Na/K ATPase inhibition by MBG and RBG reveal that these agents are equally effective as inhibitors of the enzyme.

Fulvestrant-3 Boronic Acid (ZB716): An Orally Bioavailable Selective Estrogen Receptor Downregulator (SERD).

Orally bioavailable SERDs may offer greater systemic drug exposure, improved clinical efficacy, and more durable treatment outcome for patients with ER-positive endocrine-resistant breast cancer. We report the design and synthesis of a boronic acid modified fulvestrant (5, ZB716), which binds to ERα competitively (IC50 = 4.1 nM) and effectively downregulates ERα in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. Furthermore, It has superior oral bioavailability (AUC = 2547.1 ng·h/mL) in mice, indicating its promising clinical utility as an oral SERD.

Tissue distribution of L-fucokinase in rodents.

L-fucose (fucose) is a monosaccharide normally present in mammals and is unique in being the only levorotatory sugar that can be synthesized and utilized by mammals. The metabolism of fucose is incompletely understood, but fucose can be synthesized de novo or salvaged. The utilization of fucose in the salvage pathway begins with phosphorylation by fucokinase. As part of an investigation of fucose metabolism in normal and disease states, we began an investigation of this enzyme. In this report, we present the tissue distribution of the enzyme in rat and mouse. The highest amount of activity was present in brain of both species. Some activity was found in all tissues examined (liver, kidney, heart, lung, spleen, brain, muscle, thymus, white adipose, testes, eye, aorta, small intestine, and submaxillary gland). Very low levels were found in small intestine. Varying levels in the tissues seems most likely to be the result of varying amounts of fucokinase protein as no difference in the Km values of crude enzyme could be shown. Protein-bound fucose levels were determined using the L-cysteine-phenol-sulfuric acid (CPS) assay. There is not a good correlation between fucokinase activity and protein-bound fucose, suggesting some tissues are more active in synthesis of fucose than others.

Glyceollin, a novel regulator of mTOR/p70S6 in estrogen receptor positive breast cancer.

An estimated 70% of breast cancer tumors utilize estrogen receptor (ER) signaling to maintain tumorigenesis and targeting of the estrogen receptor is a common method of treatment for these tumor types. However, ER-positive (+) breast cancers often acquire drug resistant or altered ER activity in response to anti-estrogens. Here we demonstrate glyceollin, an activated soy compound, has anti-estrogen effects in breast cancers. We demonstrate through estrogen response element luciferase and phosphorylation-ER mutants that the effects of glyceollin arise from mechanisms distinct from conventional endocrine therapies. We show that glyceollin suppresses estrogen response element activity; however, it does not affect ER-alpha (α) phosphorylation levels. Additionally we show that glyceollin suppresses the phosphorylation of proteins known to crosstalk with ER signaling, specifically we demonstrate an inhibition of ribosomal protein S6 kinase, 70 kDa (p70S6) phosphorylation following glyceollin treatment. Our data suggests a mechanism for glyceollin inhibition of ERα through the induced suppression of p70S6 and demonstrates novel mechanisms for ER inhibition.

A Ligand-Based Drug Design. Discovery of 4-Trifluoromethyl-7,8-pyranocoumarin as a Selective Inhibitor of Human Cytochrome P450 1A2.

In humans, cytochrome P450 1A2 is the major enzyme metabolizing environmental arylamines or heterocyclic amines into carcinogens. Since evidence shows that planar triangle-shaped molecules are capable of selectively inhibiting P450 1A2, 16 triangular flavone, and coumarin derivatives were designed and synthesized for these studies. Among these compounds, 7,8-furanoflavone time-dependently inhibits P450 1A2 with a K(I) value of 0.44 µM. With a 5 min preincubation in the presence of NADPH, 0.01 µM 7,8-furanoflavone completely inactivates P450 1A2 but does not influence the activities of P450s 1A1 and 1B1. Another target compound, 7,8-pyrano-4-trifluoromethylcoumarin, is found to be a competitive inhibitor, showing high selectivity for the inhibition of P450 1A2 with a K(i) of 0.39 µM, 155- and 52-fold lower than its K(i) values against P450s 1A1 and 1B1, respectively. In yeast AhR activation assays, 7,8-pyrano-4-trifluoromethylcoumarin does not activate aryl hydrocarbon receptor when the concentration is lower than 1 µM, suggesting that this compound would not up-regulate AhR-caused P450 enzyme expression. In-cell P450 1A2 inhibition assays show that 7,8-pyrano-4-trifluoromethylcoumarin decreases the MROD activity in HepG2 cells at concentrations higher than 1 µM. Thus, using 7,8-pyrano-4-trifluoromethylcoumarin, a selective and specific P450 1A2 action suppression could be achieved, indicating the potential for the development of P450 1A2-targeting cancer preventive agents.

Characterization of selective estrogen receptor modulator (SERM) activity in two triarylethylene oxybutyric acids.

Previously we identified 4-[1-(4-hydroxyphenyl)-2-phenylbuten-1-yl]phenoxy-n-butyric acid (4HBA) and its des-hydroxy analog (BA) as potential selective estrogen receptor modulators (SERMs) in the ovariectomized (OVX) rat. The aim of the present study was to characterize comprehensively the effects of 4HBA and BA in both the OVX rat and in estrogen-responsive cells. Thus, 4HBA was found to be an estrogen antagonist with partial agonist efficacy in estrogen-responsive reporter gene and estrogen-dependent proliferation assays (MVLN cells and MCF-7 human breast cancer cells, respectively). In the OVX rat, 4HBA and BA were equally effective and comparable to other known SERMs regarding (a) serum cholesterol reduction and suppression of serum markers of excessive bone metabolism, and (b) partial agonist efficacy in reproductive tissue relative to steroidal estrogens. Like steroidal estrogens, both compounds increased serum triglyceride levels, with BA being more effective in this regard. The maximal effects of 4HBA on all of these parameters except cholesterol lowering were seen at oral doses of 0.4 micromol/kg/day; maximal cholesterol lowering required doses of 10 micromol/kg/day. In OVX rat liver 9S fraction, BA was found to be efficiently converted to a single hydroxylated metabolite, 4HBA. These results suggest that the effects of BA in the OVX rat might, in part, be a consequence of biotransformation to 4HBA, and that those of 4HBA and BA in the OVX rat and in estrogen-responsive cells are qualitatively similar to those of SERMs such as tamoxifen and raloxifene.